RAG: a recombinase diversified

Abstract

During B cell and T cell development, the lymphoid-specific proteins RAG-1 and RAG-2 act together to initiate the assembly of antigen receptor genes through a series of site-specific somatic DNA rearrangements that are collectively called variable-diversity-joining (V(D)J) recombination. In the past 20 years, a great deal has been learned about the enzymatic activities of the RAG-1-RAG-2 complex. Recent studies have identified several new and exciting regulatory functions of the RAG-1-RAG-2 complex. Here we discuss some of these functions and suggest that the RAG-1-RAG-2 complex nucleates a specialized subnuclear compartment that we call the 'V(D)J recombination factory'.

Figure 1. Multilayered regulation of V(D)J recombination

Regulatory mechanisms that are RAG-dependent are shown in white panels (c, e, and f), and those that are RAG-independent are in tan (a, b, and d). a) Subnuclear localization of antigen receptor loci. Within the nucleus (yellow circle) the homologous Igh alleles are depicted in blue. Arrows indicate the centripetal movement of Igh alleles prior to initiation of V(D)J recombination at this locus; b) Sense and antisense non-coding RNA. V_H (green), D_H (red), J_H ( blue), and C_μ (orange) gene segments are shown. Right- and left-facing arrows represent sense and antisense transcription start sites, respectively; c) Allelic pairing. As in a, the nucleus is shown as a yellow circle and homologous Igh alleles are depicted in blue; d) Locus looping and contraction. The curved black line is the Igh locus, with V_H gene segments in green and rearranged DJ_H gene segments in blue. As a result of chromosomal looping, the distance between V_H gene segments and the DJ_H gene segment is decreased; e) Recognition of histone H3 methylation. A space-filling model of the RAG2 PHD finger is shown in blue, bound to the N-terminal tail of histone H3 (shown in yellow), which is trimethylated at lysine 4 and symmetrically dimethylated at arginine 2; f) Preferential usage of the classical NHEJ (cNHEJ) double-strand break repair pathway. The RAG recombinase aids in directing V(D)J recombination intermediates to cNHEJ and away from homologous recombination (HR) and alternative NHEJ (aNHEJ). Signal ends: red and yellow triangles; coding ends: U-shaped black lines.

Figure 2. Model depicting a putative V(D)J recombination factory

Within the nucleus (gray circle), the V(D)J recombination machinery may exist as a specialized, centrally-located, subnuclear compartment (yellow circle). This compartment is likely to be distinct from transcription factories (blue). An expanded view of the V(D)J recombination factory is shown on the right. Igh locus: curved black line; V_H gene segments: red, a rearranged DJ_H gene segment: blue. The RAG1-RAG2 recombinase (light purple) is shown synapsing the DJ_H gene segment and one of the V_H gene segments just prior to V_H-DJ_H recombination. Factors that are likely to be enriched within the V(D)J recombination factory are indicated. The central position of the RAG recombinase within this figure reflects the hypothesis that the RAG1-RAG2 complex nucleates the factory, while arrows pointing towards the RAG1-RAG2 complex represent the recruitment of additional regulatory factors by the RAG1-RAG2 complex.